Ubiquitin conjugation is involved in many critical biological processes, such as cell cycle control, gene transcription, and signal transduction. Dysregulation of the ubiquitin system has been implicated in major human diseases, including cancer, inflammation, and immune diseases. The long-term goal of our research is the understanding of protein ubiquitination in the regulation of immune responses. Particularly, we have studied two critical E3 ligases Itch and Cbl-b in immune regulation and have made significant progress during the current funding period, which establish these E3 ligases as the critical regulators in T cell activation, differentiation, and tolerance induction. This application of competitive renewal is based on recent novel preliminary results, which let us put forward a working hypothesis that unconventional K33- linked polyubiquitination and its reversal plays a critical role in immune regulation. We propose two Specific Aims to test this hypothesis by using a combination of biochemical, molecular, genetic, and proteomic approaches. First we plan to identify and characterize K33-linked substrates involved in the regulation of T cells. Second, we will study the process of deubiquitination in the removal of K33-linked ubiquitin chains and the biological function in T cells. The proposal will investigate the basic mechanisms of ubiquitin conjugation pathway in regulating signal transduction in the context of T cell activation and tolerance. The knowledge and outcomes obtained from this application will provide a new paradigm of the ubiquitin system in immune regulation, and at the same time, should be helpful in the rationale design of novel therapeutic approaches or intervention strategies to immunological diseases.